Ultrafast third harmonic micro-spectroscopy reveals a two-photon resonance in human hemoglobin

Abstract

The recently developed technique of ultrafast third harmonic generation (THG) micro-spectroscopy is discussed. The approach is easily adapted to a standard laser scanning microscope and allows for two and three photon resonances to be identified in non-fluorescent unlabeled samples. This work provides nonlinear microscopists with a tool for further understanding the contrast and damage mechanisms they will encounter under nonlinear excitation. Here, we use THG micro-spectroscopy to investigate the nonlinear optical properties of hemoglobin over the spectral range of 770-1000 nm with 100 fs long nJ energy laser pulses. We demonstrate the ability of this approach to distinguish different ligand binding states in physiological solutions of human hemoglobin.

Strong Anisotropy in Liquid Water upon Librational Excitation Using Terahertz Laser Fields

“…Non-equilibrium molecular dynamics simulations have shown a rapid loss of the excitation energy of a single rotationally excited water molecule in the liquid phase on the sub-100 fs time scale, where the dominant dissipation pathway involves rotation of the hydrogen-bonded molecules in the first hydration shell of a central water . This dissipation time scale is commensurate with previous experimental and theoretical studies showing that librational motions relax on a sub-200 fs time scale, − with a characteristic decay time of 75 fs. , As a result, the third-order birefringence induced in water by either static fields , or laser excitation ,− has been found to be small, and , respectively.…”

“…From the peak pump-probe measured values, we determine that the birefringent third-order response of bulk water at 12.3 THz is c 3 = (2.5 ± 0.2) • 10 &'3 )* + , + . This value is about three orders of magnitude larger than for static fields 26,27 (3.1 • 10 &'( )* + , + ) and exceeds by 5 orders of magnitude the non-linear response of pure water at optical, near-infrared 32,33,[35][36][37][55][56][57][58] , and low THz 28 frequencies, 2.6 • 10 &'/ )* + , + . As a further control we also carried out single-color pump-probe experiments at 7.8 and 13.9…”

“…This dissipation timescale is commensurate with previous experimental and theoretical studies showing that librational motions relax on a sub-200 fs timescale [18][19][20][21][22][23] , with a characteristic reorientational decay time of 75 fs 24,25 . As a result, the third-order birefringence induced in water by either static fields 26,27 or laser excitation [28][29][30][31][32][33][34][35][36][37] has been found to be small, 3.1 • 10 &'( )* + , + and 2.6 • 10 &'/ )* + , + , respectively. Here, we report single-color THz pump-THz probe experiments carried out on the ultra-bright free electron laser FELIX that provides an unprecedented degree of laser induced transient birefringence in bulk liquid water, which is found to be five orders of magnitude larger at 12.3 THz than previously reported values for excitations in the infrared, optical, and low-frequency THz ranges.…”

Towards Intense THz Spectroscopy on Water: Characterization of Optical Rectification by GaP, OH1, and DSTMS at OPA Wavelengths

“…The pump intensity required to drive liquid water into the nonlinear response regime depends on the wavelength. Based on the third-order responses reported previously, we tentatively estimate to 50 GW/cm 2 , 5 TW/cm 2 , 1 GW/cm 2 , and 5 TW/cm 2 , the peak power required to induce a pump-probe signal of roughly 1% at the frequency of~1 MHz [127,128],~1 THz [72], 10 THz [54], and~200 THz [129][130][131][132][133][134][135][136][137][138][139][140], respectively.…”

Application of P(VDF-TrFE) Glass Coating for Robust Harmonic Nanoparticles Characterization

“…This method is widely used to obtain structural information about a variety of biological specimens. Its nature allows depth-resolved imaging [ 40 ] of inhomogeneities, with practically no background from surrounding homogeneous media. With an appropriate illumination geometry, third harmonic generation microscopy is shown to be particularly suitable for imaging of biogenic components and is perspective for studying of biological tissues [ 29 , 41 ].…”